mass production of the beneficial nematode steinernema ... · nematode culture will be important as...

Mass Production of the Beneficial Nematode

Steinernema carpocapsae Using Solid State

Fermentation

Ashraf Alsaidi, Jeison Valencia, Devang Upadhyay, Sivanadane Mandjiny, Rebecca Bullard-Dillard, Jeff

Frederick, and Leonard Holmes The University of North Carolina at Pembroke, Pembroke, NC, USA

Email: [email protected]

Abstract—Steinernema carpocapsae is a microscopic

entomopathogenic nematode (EPN) that may be used as an

alternative to chemical pesticide. This species creates a

symbiotic relationship with the bacteria Xenorhabdus

nematophila. This biological control agent has many

advantages compared to chemical pesticides as it does not

harm either the environment or humans. Steinernema

carpocapsae is a vector for the bacteria to infect the targeted

insect pest. The bacteria kills the host within 24-48 hours.

This paper focuses on the mass production of beneficial

nematodes using solid state fermentation. The purpose of

the experiment was to find the optimum conditions to mass

produce the nematode efficiently. Maximizing yield with the

minimalized nutrients will increase the cost efficiency of

production, making it a more affordable attractive

alternative to harmful chemical pesticides.

Index Terms—beneficial nematode, solid state fermentation,

Steinernema carpocapsae, Xenorhabdus nematophila

I. INTRODUCTION

Steinernema carpocapsae are nematodes that evolved

a symbiotic relationship with the bacteria, Xenorhabdus

nematophila, which belongs to the family

Enterobacteriaceae. The bacteria live inside the nematode,

which serves as a vector for the bacteria into the insect

host. In return, the bacteria kill the insect by secreting

protein toxin(s) into the hemolymph and bioconverting

the insect into nutritional components for both the

nematode and the bacteria [1]. The secreted

antimicrobials are speculated to be a defense mechanism

used to ward off competing microbes [2]. S. carpocapsae

has become a center of attention for bio-ag researchers

because it is safe and can kill harmful insects within 24-

48 hours [3]. Steinernema carpocapsae “is a natural and

effective alternative to chemical pesticides, and have no

detrimental effect on non-target species” [4]. Steinernema

carpocapsae is known to attack its host by “ambushing”

migrating insects [5], [6]. The diversity of insects

affected by the biological control agent is narrow

compared to the diversity of those insects controlled by

chemical pesticides. The symbiotic pair have low heat

Manuscript received May 21, 2018; revised September 7, 2018.

tolerance and cannot survive the internal body heat of

humans [7].

The bacteria life cycle has two phenotypes, Phase I and

Phase II. Phase I bacteria are infective, whereas Phase II

are non-infective cells [8]. Reports demonstrate that

stressful conditions increase the production of stable

Phase II cultures. Mechanisms which cause phase

variation are yet to be identified [9]-[12]. Unlike

culturing nematodes in suspension where pH control is

possible, control of pH on solid state media after

inoculation is not possible. Studies of pH effects on

nematode culture will be important as this technology

continues to advance.

The purpose of this research was to mass produce the

nematode using a solid state fermentation process. Media

composition was modified to determine optimal growth

conditions. The media formulation contained nutrient

broth, beef extract, yeast extract, peptone, agar, olive oil,

and canola oil. Oil was necessary as demonstrated in

earlier studies [13]. The concentration of the media was

modified to 0.5x, 1x, 1.5x, and 2x. The nematode

inoculum concentrations were also varied. Reducing the

production cost of the beneficial nematode is as important

as having a high nematode final yield. Cheaper mass

production of the beneficial nematode will facilitate their

use as a replacement of agricultural chemical pesticides.

II. MATERIALS AND METHODS

A. Isolation of Xenorhabdus nematophila

Galleria mellonella insect larvae were used to isolate

the nematode symbiont X. nematophila [14]. Infected

larva were dead and turned dark brown color after 24

hours. The color change verified that the biocontrol agent

killed the larvae. Bacterial isolation was performed by the

method of Inman and Holmes [15]. Deceased insect

larvae were sterilized by plunging them four times into

isopropanol, followed by rinsing with sterilized water.

Air-dried, sanitized larva were dissected to isolate X.

nematophila from the hemolymph [13]. The hemolymph

was transferred onto nutrient agar bromothymol blue

tetrazolium chloride agar (NBTA) plates to differentiate

Phase I from Phase II cells. Blue colonies indicate Phase I

cells [15]. NBTA contained per liter: 8.0 g nutrient agar;

Journal of Advanced Agricultural Technologies Vol. 5, No. 4, December 2018

©2018 Journal of Advanced Agricultural Technologies 276doi: 10.18178/joaat.5.4.276-280

25 mg bromothymol blue; 40 mg 2,3,5-

triphenyltetrazolium chloride (TTC). Blue colonies were

sub-cultured and streaked onto a 2xNutrient Broth

(Carolina Biological, USA) media agar plate and

incubated at 28ºC. The bacteria were periodically

transferred onto fresh 2xNB plates throughout the study

to preserve the culture.

B. Sanitization of Steinernema carpocapsae

Steinernema carpocapsae nematodes were obtained

from Arbico Organics (Tucson, AZ. USA) and used

throughout this study. The nematodes were sanitized as

follows: Nematodes were gently shaken overnight in

sterile tap water at 150 rpm and room temperature. To

sanitize the nematodes, 0.125% Hyamine® (Sigma) was

added and incubated for 20 minutes on shaker [16]. The

sanitized nematodes were transferred into 50 mL sterile

tubes and centrifuged for 5 minutes at 500 rpm and the

pellet was collected. The process was repeated 10-12

times to remove dead bacteria from the supernatant. A

sample was collected every 3 cycles and gram stained to

check the presence of bacterial load.

C. Preparation of Solid Media

All glassware and equipment were washed thoroughly

using de-ionized water and autoclaved to remove

contamination. Fig. 1 shows the small, medium, and large

glass plates used during this experiment. The surface

areas for small, medium, and large glass plates were

approximately 400 cm2, 500 cm2 and 700 cm2

respectively, and fermentation media volumes were 400

mL, 500 mL and 700 mL respectively. The depth of solid

media bed was maintained at 1 cm in all glass plates. The

fermentation media was composed of 1% beef extract,

1% yeast extract, 2% peptone, 1% olive oil, 1% canola oil,

agar and pH was adjusted to 7.5. The media was blended

for five minutes to ensure all the ingredients were mixed

well and then poured into the plates. The loaded plates

were covered with aluminum foil and autoclaved at

121°C for 30 minutes.

Figure 1. Plates used during the experiment (large, medium, and small)

D. Inoculation of Sanitized Steinernema carpocapsae

The presence of a whitish lawn coating the media

surface indicated bacterial growth. Phase I cells of X.

nematophila were confirmed by gram staining. Surface

sanitized S. carpocapsae were then aseptically introduced

to the bacteria. The nematode inoculum concentrations

were adjusted according to the experiments. The

incubation period necessary to obtain the first generation

growth cycle of nematodes was observed on day 13 at

room temperature.

E. Harvesting and Packing the Nematodes

First generations of Steinernema carpocapsae were

harvested from the plates. Harvesting was accomplished

by adding sterile tap water on top of the solid media and

washing the nematodes by gentle shaking. The nematodes

were counted and centrifuged at 2500 rpm. The pellets

were packed in 5mm open cell sponge material and stored

at 4°C.

F. Nematode Yield

Nematode yield was determined using a gridded

sedwick rafter counting cell® (Wildco) by serial dilution

[17]. The final fold of nematodes was calculated as the

ratio of harvest concentration to inoculum concentration.

For example: 1.8 x107 nematodes (yield)/ 5.0x10

5

nematodes (inoculum) = 36 folds

III. RESULTS AND DISCUSSION

A. Effect of Nematode Inoculum Concentration on Yield

Fig. 2 shows that nematode inoculum concentrations

increase or decrease the final nematode yield. Increasing

nematode density will increase metabolic waste and

competition for food. To illustrate: inoculating 250

nematodes per cm2, resulted in 110 folds. Microscopic

examination showed the adults were larger, contained

more eggs and yielded higher production of nematodes.

When the nematode inoculum was doubled to 550

nematodes per cm2, the final folds decreased by 50%.

Johnson et al. (2016) reported 25 fold yield of the

beneficial nematode Heterorhabditis bacteriophora by

inoculating 900 nematodes per cm2 of solid media surface

which correlates closely with this report [17]. Upadhyay

et al. (2015) reported 19 folds of the entomopathogenic

nematode Heterorhabditis bacteriophora using liquid

culture fermentation technology [18]. However, in terms

of absolute production, solid state fermentation yielded

110 maximum fold, but relatively small final yield.

Liquid medium technology has the advantage of

nematodes using three dimensions, whereas solid state

fermentation is limited to two dimensions (the upper

surface of the media.)

Figure 2. Nematode yield as a function of nematode density

Few nematodes were observed within the solid media

matrix. Higher nematode density, less availability of

nutrients and more waste metabolites may have repressed


©2018 Journal of Advanced Agricultural Technologies 277

the nematodes’ ability to produce eggs. From a mass

production perspective, fold is not always the preferred

measurement of success. Another approach to

commercialization might be to shorten the ‘recovery

period’ of IJs [19], [20]. The exit from the

developmentally arrested third juvenile stage (IJ3) is

called “recovery.” Time of recovery may be important to

achieve an economically feasible production process.

Nematode recovery is dependent on various factors

including bacterial phase variant, media formulation and

bacterial density [21], [22]. Finally, nematode and

bacterial density are certainly factors affecting final

nematode yield [21], [22].

B. Effect of Fermentation Media Concentration on

Nematode Yield

In this experiment, approximately 1500 nematodes/cm2

were inoculated onto 700 cm2 plates containing different

concentrations of fermentation media. Results are

reported in Fig. 3. Final yield is related to nutrient

concentration. Doubling media concentration resulted in

decreased yield.

Figure 3. Nematode yield of Steinernema carpocapsae per gram of media ingredients/Liter

The 0.5x concentration (20 g/L) of original

fermentation media concentration was observed to

maximize final yield per gram amount of media used.

Johnson et al. reported (2016) 5.5x105 H. bacteriophora

IJs per gram of nutrients using same nutrient ingredients

of this study [17]. Whereas, Somwong and Petcharat

(2012) achieved 3.04x105, 2.45x10

5 and 2.98x10

5

IJs/gram Steinernema carpocapsae using different media

ingredients including dog food, powdered fish and

silkwarm pupa respectively in their study [23]. Fresh

chicken was used as media by Tabassum and Shahina

(2004) who reported 7.5x104 IJs/gram H. indica spp [14].

Both lipid quality and quantity affect the final nematode

yield [24]. Hence, 1% olive oil and 1% canola oil were

used in growth media throughout this study. Optimization

of lipid concentration can be conducted to obtain

maximum yield of nematodes in future studies. Nematode

production can be commercialized by optimizing culture

conditions, inoculum size, and incubation period as well

as by verifying media elements and concentrations.

To many researchers, beneficial nematodes

Heterorhabditis bacteriophora and Steinernema

carpocapsae in vitro mass production on a large scale is

challenging and cumbersome due to various obstacles

[25]. The factors responsible for making nematode mass

production difficult are: (a) phase shifting of the bacterial

symbiont; (b) concentrations of inoculum

(bacterial/nematode); (c) fermentation parameters pH,

temperature, oxygen concentration, etc. (d) aseptic

handling; (e) low percentages of nematode copulation.

Contamination is also a challenge in mass production of

nematodes. The advantage of growing X. nematophila

prior to the nematode inoculation on solid media is the

increase of secreted antimicrobial compounds, thereby

preventing contamination [26]. Success in nematode mass

production requires growing the bacterial symbiont

within the media prior to nematode inoculation [27].

Nematode yield is dependent on the concentration and

composition of media components [28]. Yoo et al.

reported that media solution containing high sources of

mono-unsaturated fatty acids and few saturated fatty

acids favor optimal growth and development of

nematodes. Yoo et al. developed a media with the

mixture of olive and canola oil for the growth of

Heterorhabditis bacteriophora. High lipid concentration

promotes long term food supply. However the bacteria

have limited ability to convert mono-unsaturated fatty

acids into usable energy [24]. In the fermentation media,

peptone was used as a principal source of organic

nitrogen and yeast/beef extracts provided amino acids,

peptides, vitamins and carbohydrates to support growth

[28].

IV. CONCLUSION

This study provides valuable guidance on

implementing a solid state fermentation technology for

mass production of S. carpocapsae. The research

demonstrates principles to achieve highest yield of

nematodes. As nematode inoculum concentration

increases, the nematode final fold decreases, though

nematode final yield increases. Finally, nematodes as a

biological control agent are important because insects

build resistance toward chemical pesticides. Not only is

the bio-control agent safe for the environment and

humans, but it does not harm beneficial insects. It is

concluded that increasing the nutrient concentration did

not benefit nematode mass production. Lowering the

nutrients may help to decrease production cost. Future

solid media studies for making production cheaper may

focus on the use of natural raw material.

ACKNOWLEDGMENT

The authors thank to Farm Bureau of Robeson County

and UNCP Department of Chemistry and Physics for

financial assistance.

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325-335, 2017.

Ashraf Alsaidi was born in Yemen and moved to the United States back in 2009. His

BS degree was in Bio-med from the University of North Carolina at Pembroke. He

had the opportunity of having a year of

Agriculture research with Dr. Holmes and Devang. He has been admitted to pharmacy

school at Wingate University starting in the fall of 2018. He hopes to have more research

opportunity in the future and hopefully teach

one day.

Jeison V Mazuera was born in Colombia,

South America in 1995. He came to America

when he was seven years old and fell in love with science because of a cartoon show called

“The Powerpuff Girls.” He went to the University of North Carolina at Pembroke.

There, he received his bachelor’s degree in

Biology and studied agriculture research for two years. He has recently been accepted to

Rush University to earn his Doctor of Philosophy. He has always been fascinated by neuroscience research but

there isn’t any type of science that doesn’t bring him enjoyment to learn

about.

Devang Upadhyay was born on December 1,

1985 in Gujarat, India. He is Biotechnology

Research and Teaching Associate in Department of Chemistry and Physics,

University of North Carolina at Pembroke, Pembroke NC, USA. He received his MS in

Microbiology from Gujarat Vidyapeeth,

Ahmedabad, India in 2009, and BS in Microbiology from Gujarat Vidyapeeth,

Ahmedabad, India in 2007. His research

interests include fermentation microbiology, Biofuel, Bioenergy,

Applied Science, biological processes, and Bio-Ag.



Sivanadane Mandjiny was born on September 14, 1955 in Pondicherry, India.

Presently he is Professor of Chemistry and

Chair in Department of Chemistry and Physics, University of North Carolina at Pembroke,

Pembroke NC, USA. He received his B.Tech. in Chemical Engineering from University of

Madras, Madras, India in 1979, M. Tech. in

Biochemical Engineering from Indian Institute of Technology, New Delhi, India in 1981,

M.Eng. in Chemical Engineering from University of Toronto, Toronto, Canada in 1984, and Ph.D. in Biological Engineering from Universite

de Technologie de Compiegne, France in 1992. His research interests

include Biomanufacturing, Biofermentation, Bioenergy, and Bioengineering.

Rebecca Bullard-Dillard was born on

September 20, 1959 in Sylacauga, Alabama,

United States. She received her Ph.D. (Biochemistry emphasis) from University of

South Carolina, Chemistry in 1996, and B.S. (cum laude) from North Carolina State

University, Biochemistry in 1990. Now she is

Associate Vice Chancellor for Research and Sponsored Programs and Professor of

Chemistry, in University of North Carolina at Pembroke, Pembroke NC, USA. Her research interests include

Biomanufacturing, Biofermentation, Bioenergy, and Bioengineering.

Jeff Frederick was born on December 9, 1963. He received his Ph.D. in American History

from Auburn University in 2003, MA in

History from University Central Florida, and B.S.B.A. in Marketing from University

Central Florida. He is Dean in College of Arts and Sciences. His research interests include

the narrative and interpretation of twentieth-

century southern politics and its impact on society, The interplay of race, class, and

gender in the transformation of the post-World War II South, Professional, collegiate, and recreational sports as a mirror of American

cultural values, and Strategies and tactics for student development and

success.

Leonard Holmes was born on May 3, 1948.

He is Associate Professor in Department of

Chemistry and Physics, University of North Carolina at Pembroke, Pembroke NC, USA.

He received his BS in Biology in 1981 from Westfield state College, Westfield, MA, and

Ph.D. in Biochemistry in 1989 from Utah

State University, Logan, UT. His research interests include fermentation microbiology,

Applied Science, biological processes, and Bio-Ag.



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